Evolution of bacteriophage tails: Structure of T4 gene product 10

The success of tailed bacteriophages to infect cells far exceeds that of most other viruses on account of their specialized tail and associated baseplate structures. The baseplate protein gene product (gp) 10 of bacteriophage T4, whose structure was determined to 1.2 A resolution, was fitted into the cryo-electron microscopy structures of the pre and post-infection conformations of the virus. gp10 functions as a molecular lever that rotates and extends the hinged short tail fibers to facilitate cell attachment. The central folding motif of the gp10 trimer is similar to that of the baseplate protein gp11 and to the receptor-binding domain of the short tail fiber, gp12. The three proteins comprise the periphery of the baseplate and interact with each other. The structural and functional similarities of gp10, gp11, and gp12 and their sequential order in the T4 genome suggest that they evolved separately, subsequent to gene triplication from a common ancestor. Such events are usual in the evolution of complex organelles from a common primordial molecule.     

Structure and location of gene product 8 in the bacteriophage T4 baseplate

Many bacteriophages, such as T4, T7, RB49, and phi29, have complex, sometimes multilayered, tails that facilitate an almost 100% success rate for the viral particles to infect host cells. In bacteriophage T4, there is a baseplate, which is a multiprotein assembly, at the distal end of the contractile tail. The baseplate communicates to the tail that the phage fibers have attached to the host cell, thereby initiating the infection process. Gene product 8 (gp8), whose amino acid sequence consists of 334 residues, is one of at least 16 different structural proteins that constitute the T4 baseplate and is the sixth baseplate protein whose structure has been determined. A 2.0A resolution X-ray structure of gp8 shows that the two-domain protein forms a dimer, in which each monomer consists of a three-layered beta-sandwich with two loops, each containing an alpha-helix at the opposite sides of the sandwich. The crystals of gp8 were produced in the presence of concentrated chloride and bromide ions, resulting in at least 11 halide-binding sites per monomer. Five halide sites, situated at the N termini of alpha-helices, have a protein environment observed in other halide-containing protein crystal structures. The computer programs EMfit and SITUS were used to determine the positions of six gp8 dimers within the 12A resolution cryo-electron microscopy image reconstruction of the baseplate-tail tube complex. The gp8 dimers were found to be located in the upper part of the baseplate outer rim. About 20% of the gp8 surface is involved in contacts with other baseplate proteins, presumed to be gp6, gp7, and gp10. With the structure determination of gp8, a total of 53% of the volume of the baseplate has now been interpreted in terms of its atomic structure.     

The wac gene product of bacteriophage T4 contains coiled-coil structural patterns

The bacteriophage T4 late gene wac (whisker antigen control) encodes the protein which forms the fibrous structure on the neck of the virion called whiskers. Amino acid sequence analysis of wac gene product, as deduced from the nucleotide sequence, indicate ten alpha-helical domains (19-40 residues long) with coiled-coil structural patterns. These regions comprise about 70% of the entire 486 amino acid sequence. The alpha-helices are separated by short stretches of polypeptide chain which are similar to the loop regions of the globular protein sequences. We propose a structural model for the dimer of wac gene product molecule, that we call fibritin in which two polypeptide chains associate in a parallel fashion and form a segmented alpha-helical coiled-coil rod similar to epidermal keratins.     

The structure of bacteriophage T4 gene product 9: the trigger for tail contraction.

BACKGROUND: The T4 bacteriophage consists of a head, filled with double-stranded DNA, and a complex contractile tail required for the ejection of the viral genome into the Escherichia coli host. The tail has a baseplate to wh?ch are attached six long and six short tail fibers. These fibers are the sensing devices for recognizing the host. When activated by attachment to cell receptors, the fibers cause a conformational transition in the baseplate and subsequently in the tail sheath, which initiates DNA ejection. The baseplate is a multisubunit complex of proteins encoded by 15 genes. Gene product 9 (gp9) is the protein that connects the long tail fibers to the baseplate and triggers the tail contraction after virus attachment to a host cell. RESULTS: The crystal structure of recombinant gp9, determined to 2.3 A resolution, shows that the protein of 288 amino acid residues assembles as a homotrimer. The monomer consists of three domains: the N-terminal domain generates a triple coiled coil; the middle domain is a mixed, seven-stranded beta sandwich with a topology not previously observed; and the C-terminal domain is an eight-stranded, antiparallel beta sandwich having some resemblance to 'jelly-roll' viral capsid protein structures. CONCLUSIONS: The biologically active form of gp9 is a trimer. The protein contains flexible interdomain hinges, which are presumably required to facilitate signal transmission between the long tail fibers and the baseplate. Structural and genetic analyses show that the C-terminal domain is bound to the baseplate, and the N-terminal coiled-coil domain is associated with the long tail fibers.     

Mutational analysis of the phage T4 morphogenetic 31 gene, whose product interacts with the Escherichia coli GroEL protein

The phage T4 morphogenetic gene 31 has been sequenced. Its deduced gene product is a polypeptide of 111 aa, with a predicted Mr of 12064 and a pI of 4.88. The proof that the assigned open reading frame (ORF) encodes Gp31 rests on the sequencing of two known gene 31 amber mutations, amN54 and NG71, demonstrating that these mutations result in translational termination within the assigned ORF. Furthermore, the sequencing of four different T4 epsilon mutations, isolated on the basis of allowing the phage to propagate on Escherichia coli groEL- hosts, showed that they are either missense mutations or 3-bp deletions in the gene 31 reading frame. The sequencing of neighboring DNA revealed the presence of five other ORFs, one of which overlaps gene 31 substantially, but in the opposite orientation.     

Analysis of expression of the rII gene function of bacteriophage T4.

Temperature-sensitive (ts) mutants of the T4 phage rII gene were islated and used in temperature shift experiments that revelaed two different expressions for the normal rII (rII+) gene function in vivo: (i) an early expression (0 to 12 min postinfection at 30 C) that prevents restriction of T4 growth in Escherichia coli hosts lysogenic for gamma phage, and (ii) a later expression (12 to 18 min postinfection at 30 C) that results in restriction of T4 growth when the phage DNA ligase (gene 30) is missing. The earlier expression appeared to coincide with the period of synthesis of the protein product of the T4 rIIA cistron, whereas the later expression occurred after rIIA protein synthesis had stopped. The synthesis of the protein product of the rIIB cistron continues for several minutes after rIIA protein synthesis ceases (O'Farrell and Gold, 1973). The two rII+ gene expressions might require different molar ratios of the rIIA and rIIB proteins. It is possible that the separate expressions of rII+ gene function are manifestations of different associations between the two rII proteins and other T4-induced proteins that are synthesized or activated at different times after phage infection.     

Partial exclusion of bacteriophage T2 by T4: An early function of the second linkage group

Evidence for exclusion as an early function of phage T4 was obtained along the following lines: a) In crosses of T2 and T4am 122, a mutant of an early phage enzyme, T2 is virtually eliminated from the progeny; b) there is no net synthesis of DNA in the mixed complexes of the non-permissive host; furthermore, there is a loss of DNA, not found in the monocomplexes of T4am 122; this suggests a specific breakdown of T2 DNA; c) a cross was made between T4 and a partially non-excludable, otherwise T2-like phage strain that did not exclude standard type T2. The gene responsible for exclusion segregated in an almost normal way and appeared to reside in-betweenh ⁺ andr at the beginning of the map segment of T4 for the early functions.The frequencies with which the genes of T2 were recovered in the progeny of this cross showed polarity, i.e. the frequency of the T2 genes along the segment of the early functions increased gradually from 0.26 for the adsorption properties to 0.44 for the UV sensitivity of T2 in correlation with the position of the genes on the map.There is a stronger effect of exclusion and a more pronounced polarity of the frequencies of recovered genes of T2 whenE. coli CR 63 is used as a host instead ofE. coli B.This work was carried out partially within the frame of the association between Euratom and the University of Leiden embodied in contract nr. 052-64-1 BIAN.     

The product of the split sunY gene of bacteriophage T4 is a processed protein.

The sunY gene of bacteriophage T4 contains a self-splicing group I intron. The ligated exons encode an open reading frame of 605 amino acids, whose inferred molecular mass is 68 kDa. However, none of the proteins made following T4 infection have been assigned to the sunY gene, and no mutations have been mapped to this locus. We show here that the primary product of the sunY gene is a protein with an apparent molecular mass of 64 kDa, which is processed to a protein approximately 4 kDa smaller. Unlike most other processed T4 proteins, cleavage occurs independently of both the T4 processing protease, the product of gene 21, and late phage protein synthesis. Insertional mutagenesis demonstrated that the sunY protein is not necessary for normal T4 growth under the conditions tested.